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PubMed1992

The biosynthesis of membrane and soluble plastidic c-type cytochromes of Chlamydomonas reinhardtii is dependent on multiple common gene products.

Cytochrome c6 functions in the thylakoid lumen to catalyze electron transfer from reduced cytochrome f of the cytochrome b6f complex to P700+ of photosystem I. The biogenesis of mature cyt c6 from cytosolically translated pre-apocytochrome c6 involves numerous post-translational modifications including the proteolytic removal of a transit sequence and the covalent attachment of heme to two cysteinyl thiols on the apoprotein. Here, we report on the characterization of a previously unrecognized class of non-allelic mutants of Chlamydomonas reinhardtii that are blocked at the conversion of apocyt c6 to holocyt c6. The mutants are acetate requiring since they are also deficient in cyt f, cyt b and the Rieske FeS protein. Pulse-chase studies indicate that heme attachment is not required for the two-step processing of pre-apocytochrome c6 to apocyt c6, but is required for the stability of the mature protein. This is in contrast to the biosynthesis of mitochondrial cyt c1 where heme attachment is required for the second processing step. We propose that the assembly of both holocytochrome c6 and the cytochrome b6f complex are dependent on common gene products, possibly those involved in heme delivery or metabolism. This is the first suggestion that multiple loci are involved in the biosynthesis of both plastidic c-type cytochromes.

Animals↗

PubMed1999

Proton to electron stoichiometry in electron transport of spinach thylakoids

According to the concept of the Q-cycle, the H+/e- ratio of the electron transport chain of thylakoids can be raised from 2 to 3 by means of the rereduction of plastoquinone across the cytochrome b6f complex. In order to investigate the H+/e- ratio we compared stationary rates of electron transport and proton translocation in spinach thylakoids both in the presence of the artificial electron acceptor ferricyanide and in the presence of the natural acceptor system ferredoxin+NADP. The results may be summarised as follows: (1) a variability of the H+/e- ratio occurs with either acceptor. H+/e- ratios of 3 (or even higher in the case of the natural acceptor system, see below) are decreased towards 2 if strong light intensity and low membrane permeability are employed. Mechanistically this could be explained by proton channels connecting the plastoquinol binding site alternatively to the lumenal or stromal side of the cytochrome b6f complex, giving rise to a proton slip reaction at high transmembrane DeltapH. In this slip reaction protons are deposited on the stromal instead of the lumenal side. In addition to the pH effect there seems to be a contribution of the redox state of the plastoquinone pool to the control of proton translocation; switching over to stromal proton deposition is favoured when the reduced state of plastoquinone becomes dominant. (2) In the presence of NADP a competition of both NADP and oxygen for the electrons supplied by photosystem I takes place, inducing a general increase of the H+/e- ratios above the values obtained with ferricyanide. The implications with respect to the adjustment of a proper ATP/NADPH ratio for CO2 reduction are discussed.

Journal Article↗

PubMed1995

Biosynthesis of cytochrome f in Chlamydomonas reinhardtii: analysis of the pathway in gabaculine-treated cells and in the heme attachment mutant B6.

Chlamydomonas reinhardtii uses two c-type cytochromes for photosynthetic electron transfer: the thylakoid membrane-bound cytochrome f of the cytochrome b6f complex and the soluble cytochrome c6. Previously, a class of photosynthesis-minus, acetate-requiring mutants was identified which were deficient in both c-type cytochromes, and biochemical analyses of cytochrome c6 biosynthesis in these strains indicated that they were each blocked at the step of heme attachment to apocytochrome c6. In order to demonstrate that the deficiency in cytochrome f results from the same biochemical and genetic defect, cytochrome f biosynthesis was examined in the B6 mutant (a representative of this phenotypic class) and in spontaneous suppressor strains derived from B6. Pulse-radiolabeling experiments show that B6 synthesizes a form of cytochrome f that is rapidly degraded in vivo. This polypeptide is membrane associated and migrates with an electrophoretic mobility identical to that of standard apocytochrome f produced in vitro but slightly greater than that of standard holocytochrome f produced in vivo by wild-type cells. These findings suggest that the B6 strain is unable to convert apocytochrome f to holocytochrome f and that apocytochrome f is unstable in vivo. In the suppressed strains, accumulation of both holocytochrome f and holocytochrome c6 is restored. One suppressor mutation (strain B6R) displays uniparental inheritance whereas another (B6T3) displays Mendelian inheritance. In both cases, the three phenotypes, photosynthesis-plus, b6f+ and cyt c6+ co-segregate in genetic crosses. This study therefore confirms that the dual cyt b6f-/cytc6- deficiency in B6 results from a single mutation that affects a step in holocytochrome formation that is common to the biosynthetic pathways of both plastidic c-type cytochromes. The study also confirms that pre-apocytochrome f synthesis, processing and association with the membrane is not dependent on heme attachment. Synthesis of cytochrome f does, however, appear to be dependent on heme availability. In cells depleted of tetrapyrrole pathway intermediates by gabaculine treatment, cytochrome f synthesis was significantly reduced. Since gabaculine treatment did not affect the stability of cytochrome f nor the accumulation of cytochrome f-encoding transcripts, the reduction is attributed to post-transcriptional regulation of preapocytochrome f synthesis via a pathway that is sensitive to the availability of heme or a tetrapyrrole pathway intermediate.

Animals↗

PubMed1994

Membrane association of cytochrome b6f subunits. The Rieske iron-sulfur protein from Chlamydomonas reinhardtii is an extrinsic protein.

The mode of membrane attachment of five subunits from Chlamydomonas reinhardtii cytochrome b6f complex has been studied using biochemical approaches. Antisera specific for cytochrome f, cytochrome b6, the Rieske iron-sulfur protein, subunit IV, and a 4-kDa subunit (product of the petG gene) were used to quantify the degree of extraction of each of these polypeptides following various treatments. In contrast to the other four subunits, the Rieske protein was extracted to extents varying between 50 and 100% following two cycles of freezing and thawing in the presence of chaotropic agents (KSCN, urea, or NaI). The Rieske protein was not extracted by 2 M NaCl and was rather resistant to alkaline treatments, being extracted by 20 mM 3-(cyclohexylamino)propanesulfonic acid buffer only at pH > 11.5. The hydrodynamic behavior of the isolated Rieske protein was examined in the absence and presence of detergent by ultracentrifugation and by molecular sieving. The extracted protein bound neither to laurylmaltoside nor to C12E8 micelles. Its sedimentation coefficient (D20,w = 9.6 x 10(-11) m2 x s-1), diffusion coefficient (s20,w = 2S), an deduced molecular mass (20.0 +/- 1.7 kDa) are those expected for the monomeric protein. We conclude that the Rieske protein is extrinsic and therefore does not cross the membrane, although its association with the rest of the complex involves primarily hydrophobic interactions, and that the other four subunits analyzed are intrinsic.

Animals↗

PubMed1997

Cyclic electron transfer in Heliobacillus mobilis involving a menaquinol-oxidizing cytochrome bc complex and an RCI-type reaction center.

Flash-induced absorption changes arising from b-type hemes were studied on whole cells of Heliobacillus mobilis under physiological and redox-controlled conditions. The sensitivity of the monitored redox changes to inhibitors of cytochrome bc complexes and the redox potential dependence of reduction and oxidation reactions of cytochrome b-hemes demonstrate that the respective b-hemes are part of a cytochrome bc complex. Both the half-time and the extent of flash-induced reduction of cytochrome b titrated with apparent potentials of about -60 and -50 mV (both n = 2), respectively, i.e., close to the Em,7 value of the menaquinone (MK) pool, indicating a collisional interaction between menaquinol and the Qo site of the cytochrome bc complex. At strongly reducing ambient potentials (< -150 mV), a net flash-induced oxidation of b-hemes was observed in agreement with the Em,7 values of the individual hemes of -90 mV (b(h)) and -190 mV (b(l)) determined in equilibrium redox titrations on membrane fragments. From the extent of photooxidized b- and c-type hemes as well as P798+, a stoichiometry of 0.6-0.75 cytochrome bc complexes per photosynthetic reaction center was estimated. The kinetic behavior and also the energy profiles for Q-cycle turnover of the heliobacterial complex are compared to those of cytochrome bc1 complexes from purple bacteria and of cytochrome b6f complexes from chloroplasts.

Anaerobiosis↗

PubMed1999

TAKs, thylakoid membrane protein kinases associated with energy transduction.

The phosphorylation of proteins within the eukaryotic photosynthetic membrane is thought to regulate a number of photosynthetic processes in land plants and algae. Both light quality and intensity influence protein kinase activity via the levels of reductants produced by the thylakoid electron transport chain. We have isolated a family of proteins called TAKs, Arabidopsis thylakoid membrane threonine kinases that phosphorylate the light harvesting complex proteins. TAK activity is enhanced by reductant and is associated with the photosynthetic reaction center II and the cytochrome b6f complex. TAKs are encoded by a gene family that has striking similarity to transforming growth factor beta receptors of metazoans. Thus thylakoid protein phosphorylation may be regulated by a cascade of reductant-controlled membrane-bound protein kinases.

Activin Receptors, Type I↗

PubMed2006

Prediction of transmembrane helix orientation in polytopic membrane proteins.

BACKGROUND: Membrane proteins compose up to 30% of coding sequences within genomes. However, their structure determination is lagging behind compared with soluble proteins due to the experimental difficulties. Therefore, it is important to develop reliable computational methods to predict structures of membrane proteins. RESULTS: We present a method for prediction of the TM helix orientation, which is an essential step in ab initio modeling of membrane proteins. Our method is based on a canonical model of the heptad repeat originally developed for coiled coils. We identify the helical surface patches that interface with lipid molecules at an accuracy of about 88% from the sequence information alone, using an empirical scoring function LIPS (LIPid-facing Surface), which combines lipophilicity and conservation of residues in the helix. We test and discuss results of prediction of helix-lipid interfaces on 162 transmembrane helices from 18 polytopic membrane proteins and present predicted orientations of TM helices in TRPV1 channel. We also apply our method to two structures of homologous cytochrome b6f complexes and find discrepancy in the assignment of TM helices from subunits PetG, PetN and PetL. The results of LIPS calculations and analysis of packing and H-bonding interactions support the helix assignment found in the cytochrome b6f structure from green alga but not the assignment of TM helices in the cyanobacterium b6f structure. CONCLUSION: LIPS calculations can be used for the prediction of helix orientation in ab initio modeling of polytopic membrane proteins. We also show with the example of two cytochrome b6f structures that our method can identify questionable helix assignments in membrane proteins. The LIPS server is available online at http://gila.bioengr.uic.edu/lab/larisa/lips.html.

Algorithms↗

PubMed1997

Light-stimulated degradation of an unassembled Rieske FeS protein by a thylakoid-bound protease: the possible role of the FtsH protease.

Unassembled subunits of the cytochrome b6f complex as well as components of other unassembled chloroplastic complexes are rapidly degraded within the organelle. However, the mechanisms involved in these proteolytic processes are obscure. When the Rieske FeS protein (RISP) is imported into isolated chloroplasts in vitro, some of the protein does not property assemble with the cytochrome complex, as determined by its sensitivity to exogenous protease. When assayed in intact, lysed, or fractionated chloroplasts, the imported RISP was found to be sensitive to endogenous proteases as well. The activity responsible for degradation of the unassembled protein was localized to the thylakoid membrane and characterized as a metalloprotease requiring zinc ions for its activity. The degradation rate was stimulated by light, but no involvement of ATP or redox control was observed. Instead, when the RISP that was attached to thylakoid membranes was first illuminated on ice, degradation proceeded in either light or darkness at equal rates suggesting a light-induced conformational change making the protein prone to degradation. Antibodies raised against native FtsH, a bacterial, membrane-bound, ATP-dependent, zinc-stimulated protease, effectively inhibited degradation of the unassembled RISP, suggesting a role for the chloroplastic FtsH in this process.

Adenosine Triphosphatases↗

PubMed2004

The central role of the green alga Chlamydomonas reinhardtii in revealing the mechanism of state transitions.

This review focuses on the essential role played by the green alga Chlamydomonas reinhardtii in revealing both the mechanism and the physiological consequences of state transitions. Two aspects are considered. The first is the role of the cytochrome b6f complex in regulating state transitions, in light of the recently obtained 3D structure. The second is the switch between linear and cyclic electron flow that follows state transitions in Chlamydomonas. Structural and dynamic elements that might be involved in such a switch, as well as its consequences on the energetic metabolism, are discussed.

Animals↗

PubMed1991

Chloroplast Rieske Center. EPR study on its spectral characteristics, relaxation and orientation properties.

The spectral parameters, the orientation of the g tensor in two-dimensionally ordered multilayers and the relaxation properties of the Rieske center in purified cytochrome b6f complex from spinach were studied by EPR spectroscopy. A trough at g = 1.76 - 1.74 was unambiguously identified as the gx signal on the basis of the sensitivity of its field position to inhibitors and oxidized plastoquinone. In contrast to previous reports, the orientation of the chloroplast Rieske center was found to be identical to that of its cytochrome bc1 counterpart, provided that non-saturating EPR conditions were applied. Upon onset of microwave saturation, however, the orientation with respect to the ordered multilayers where the gy signal (g = 1.9) was maximal, changed drastically and was then similar to the orientation reported previously (Prince R. C., Crowder, M. S., and Bearden, A. J. (1980) Biochim. Biophys. Acta 592, 323-337). The unusually anisotropic saturation behavior of the EPR spectrum was characterized in detail, whereby hitherto controversial results on the spectral parameters of the chloroplast Rieske center could be rationalized. The observed phenomena are discussed on the basis of three tentative models which are able to qualitatively explain the effects.

Chloroplasts↗

PubMed2004

Photosynthetic architecture differs in coastal and oceanic diatoms.

Diatoms are a key taxon of eukaryotic phytoplankton and a major contributor to global carbon fixation. They are ubiquitous in the marine ecosystem despite marked gradients in environmental properties, such as dissolved iron concentrations, between coastal and oceanic waters. Previous studies have shown that offshore species of diatoms and other eukaryotic algae have evolved lower iron requirements to subsist in iron-poor oceanic waters, but the biochemical mechanisms responsible for their decreased iron demand are unknown. Here we show, using laboratory-cultured model species, a fundamental difference between a coastal and an oceanic diatom in their photosynthetic architecture. Specifically, the oceanic diatom had up to fivefold lower photosystem I and up to sevenfold lower cytochrome b6f complex concentrations than a coastal diatom. These changes to the photosynthetic apparatus markedly decrease the cellular iron requirements of the oceanic diatom but not its photosynthetic rates. However, oceanic diatoms might have also sacrificed their ability to acclimate to rapid fluctuations in light intensity--a characteristic of dynamic and turbid coastal waters. We suggest that diatoms, and probably other eukaryotic algal taxa, exploited this difference in the underwater light climate between oceanic and coastal waters, enabling them to decrease their iron requirements without compromising photosynthetic capacity. This adaptation probably facilitated the colonization of the open ocean by diatoms, and contributes to their persistence in this iron-impoverished environment.

Acclimatization↗

PubMed2006

Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling.

Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process. The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of magnesium protoporphyrin IX methyl ester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the magnesium chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step.

Arabidopsis↗

PubMed2005

Three maize leaf ferredoxin:NADPH oxidoreductases vary in subchloroplast location, expression, and interaction with ferredoxin.

In higher plants, ferredoxin (Fd):NADPH oxidoreductase (FNR) catalyzes reduction of NADP+ in the final step of linear photosynthetic electron transport and is also implicated in cyclic electron flow. We have identified three leaf FNR isoenzymes (LFNR1, LFNR2, and LFNR3) in maize (Zea mays) chloroplasts at approximately equivalent concentrations. Fractionation of chloroplasts showed that, while LFNR3 is an exclusively soluble enzyme, LFNR1 is only found at the thylakoid membrane and LFNR2 has a dual location. LFNR1 and LFNR2 were found to associate with the cytochrome b6f complex following its partial purification. We cloned LFNR3 and produced all three isoenzymes as stable, soluble proteins. Measurement of Fd reduction ability showed no significant differences between these recombinant enzymes. Column chromatography revealed variation between the interaction mechanisms of LFNR1 and LFNR2 with Fd, as detected by differential dependence on specific intermolecular salt bridges and variable sensitivity of interactions to changes in pH. A comparison of LFNR transcripts in leaves of plants grown on variable nitrogen regimes revealed that LFNR1 and LFNR2 transcripts are relatively more abundant under conditions of high demand for NADPH. These results are discussed in terms of the functional differentiation of maize LFNR isoenzymes.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequence of the petB gene encoding cytochrome b6 from the mesophilic cyanobacterium Synechocystis PCC 6803: implications for evolution and function.

The gene encoding the cytochrome b6 subunit (petB) of the cytochrome b6f complex has been isolated, cloned and sequenced by nonradioactive methods from genomic DNA of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 666 nucleotides, coding for a polypeptide with a molecular mass of 25.02 kDa. In contrast to higher plant petB sequences an aminoterminal extension of seven amino acids occurs. Aminoterminal sequencing of the isolated protein excludes--different from higher plants--the existence of an intron after the first amino acids but indicate the posttranslational removal of three amino acids from the amino terminus. The aminoterminal extension--found only in non-nitrogen-fixing, unicellular cyanobacteria--shows a high degree of homology between different species.

Amino Acid Sequence↗

PubMed2006

Cyclic electron flow in C3 plants.

This paper summarized our present view on the mechanism of cyclic electron flow in C3 plants. We propose that cyclic and linear pathways are in competition for the reoxidation of the soluble primary PSI acceptor, Ferredoxin (Fd), that freely diffuses in the stromal compartment. In the linear mode, Fd binds ferredoxin-NADP-reductase and electrons are transferred to NADP+ and then to the Benson and Calvin cycle. In the cyclic mode, Fd binds a site localized on the stromal side of the cytochrome b6f complex and electrons are transferred to P700 via a mechanism derived from the Q-cycle. In dark-adapted leaves, the cyclic flow operates at maximum rate, owing to the partial inactivation of the Benson and Calvin cycle. For increasing time of illumination, the activation of the Benson and Calvin cycle, and thus, that of the linear flow, is associated with a subsequent decrease in the rate of the cyclic flow. Under steady-state conditions of illumination, the contribution of cyclic flow to PSI turnover increases as a function of the light intensity (from 0 to approximately 50% for weak to saturating light, respectively). Lack of CO2 is associated with an increase in the efficiency of the cyclic flow. ATP concentration could be one of the parameters that control the transition between linear and cyclic modes.

Chlorophyll↗

PubMed2001

A comprehensive phylogenetic analysis of Rieske and Rieske-type iron-sulfur proteins.

The Rieske iron-sulfur center consists of a [2Fe-2S] cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs. Two protein families possessing Rieske centers have been defined. The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems. The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins. Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases. Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor. Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins. The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above.

Amino Acid Motifs↗

PubMed2004

Rapid evolution of RNA editing sites in a small non-essential plastid gene.

Chloroplast RNA editing proceeds by C-to-U transitions at highly specific sites. Here, we provide a phylogenetic analysis of RNA editing in a small plastid gene, petL, encoding subunit VI of the cytochrome b6f complex. Analyzing representatives from most major groups of seed plants, we find an unexpectedly high frequency and dynamics of RNA editing. High-frequency editing has previously been observed in plastid ndh genes, which are remarkable in that their mutational inactivation does not produce an obvious mutant phenotype. In order to test the idea that reduced functional constraints allow for more flexible evolution of RNA editing sites, we have created petL knockout plants by tobacco chloroplast transformation. We find that, in the higher plant tobacco, targeted inactivation of petL does not impair plant growth under a variety of conditions markedly contrasting the important role of petL in photosynthesis in the green alga Chlamydomonas reinhardtii. Together with a low number of editing sites in plastid genes that are essential to gene expression and photosynthetic activity, these data suggest that RNA editing sites may evolve more readily in those genes whose transitory loss of function can be tolerated. Accumulated evidence for this 'relative neutrality hypothesis for the evolution of plastid editing sites' is discussed.

Base Sequence↗

PubMed1994

Characterization of the gene of the chloroplast Rieske iron-sulfur protein in Chlamydomonas reinhardtii. Indications for an uncleaved lumen targeting sequence.

The sequence of the nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome b6f complex of Chlamydomonas reinhardtii has been established. Comparison of genomic clones and amplified cDNA indicates that the petC gene is interrupted by four introns within the coding sequence of the mature protein. The nucleotide sequence predicts a precursor protein of 206 amino acid residues with a transit peptide of 29 amino acids. The transit peptide is shorter than that of higher plants and has a basic region typical for the transfer through the chloroplast envelope, but no hydrophobic segment at the C-terminal end as is found in proteins transferred through the thylakoid membrane. The mature protein shows a high degree of homology with that of higher plants and has an N-terminal hydrophobic segment as in other Rieske proteins. Biochemical data (Breyton, C., de Vitry, C., and Popot, J.-L. (1994) J. Biol. Chem. 269, 7597-7602) indicate that the chloroplast Rieske protein of C. reinhardtii is an extrinsic membrane protein. Therefore, this N-terminal hydrophobic segment is not a transmembrane segment but may act as an uncleaved N-terminal thylakoid membrane transfer signal sequence. There are other examples of uncleaved hydrophobic membrane transfer signal in secreted proteins, although these are rare.

Amino Acid Sequence↗